The broad goal of this application is to gain an understanding of the contribution of osmotic loading in mediating chondrocyte behavior in culture studies of chondrocyte mechanotransduction as well as in physiologic joint loading. While this basic science study is intended to provide new information regarding chondrocyte mechanotransduction, we anticipate that some findings may shed light on the role that changes to the osmotic environment play in osteoarthritis (OA). In this A1 revised proposal, based on our preliminary data, we hypothesize that the cytoskeleton plays a critical role in mediating the osmotic loading response of articular chondrocytes. In this scenario, the cytoskeleton may be responsive to osmotic loading-induced Ca2+ changes, or the osmotic loading-induced Ca2+ changes may be responsive to the initial organization of the cytoskeleton, or both, may be operative in a feedback loop. Reported zonal differences between the cytoskeletal protein content of chondrocytes, as well as cytoskeletal differences between normal and OA chondrocytes, provide added motivation for our hypothesis. To test this hypothesis, we propose several specific aims to initiate our studies of the role that the cytoskeleton plays in mediating changes to aggrecan gene expression. Specific Aim 1a. Determine the effect of initial culture osmolarity (chronic 2 hour exposure) on cytoskeletal organization (including spatial distribution and steady-state polymer levels of microfilaments (MFs) and microtubules (MTs), total protein content of actin and tubulin, and organization and content of stable microtubules) in the cell. Specific Aim 1b. Measure the response induced by real-time osmotic loading in intracellular calcium ([Ca2+]i) (peak magnitude, percentage of responding cells) and cell size change (equilibrium volume, rate of change and material properties) of middle zone chondrocytes (MZCs) to 10 min of osmotic loading (variable magnitude and frequency) following a 2 hr pretreatment in medium of varying osmolarities. Specific Aim 1c. Repeat selected real-time [Ca2+]i studies of Specific Aim 1b on cells pretreated with pharmacological agents that modulate cytoskeletal organization or calcium mobilization. Specific Aim 2. Repeat Specific Aim 1 on superficial (SZC) and deep zone (DZC) chondrocytes. Specific Aim 3. Following a 2 hour preincubation in culture medium of varying osmolarities, we will subject chondrocytes to 2 hours of osmotic loading (variable magnitude and frequency) and we then propose: Specific Aim 3a. For SZC, MZC, and DZCs, quantify cytoskeletal organization in the presence and absence of pharmacological agents that disrupt calcium mobilization. Specific Aim 3b. Measure aggrecan gene expression after this period. Specific Aim 3c. Determine if aggrecan gene expression in response to osmotic loading is dependent on [Ca2+]i and cytoskeletal organization of actin and microtubules using a strategy of pharmacological agents that modulate cytoskeletal organization or calcium mobilization.